Humans have two genes that encode acetylcholine—hydrolyzing enzymes, AChE and BChE (Soreq and Zakut, 1993). The AChE and BChE genes, although drastically different from each other in base composition, are thought to be derived from a common ancestral gene. AChE, mapped to chromosome 7q22 encodes the primary enzyme, acetylcholinesterase (AChE, E.C. 3.1.1.7), which terminates neurotransmission at synapses and neuromuscular junctions. BuChE, mapped to 3q26 encodes butyrylcholinesterase (BChE, E.C. 3.1.1.8), a homologous serum esterase with somewhat broader substrate specificity. BuChE acts as a scavenger of natural and man-made poisons, including organophosphate and carbamate pesticides, that are increasingly a threat to human health (Loewenstein et al., 1993). Yet, individuals with no BuChE activity (silent phenotype) in their serum are apparently healthy.
AChE acquires heterogeneous properties in different tumors distinct from those it displays in muscle and nerve, hemopoietic cells, embryonic tissue and germ cells. Monomers of the catalytic AChE subunit were observed in meningiomas and tetrameres in glioblastomas. Inhibition properties different from those of normal AChE were determined for serum AChE in various carcinomas. Moreover, tumorigenic expression of the corresponding AChE gene was found to be subject to variable control mechanisms. In differentiating neuroblastoma cells, inhibition of mevallonate synthesis, which decreases proliferation rates, increases AChE levels. In PC12 cells, in contrast, nerve growth factor induces the production of hydrophilic AChE, while embryonal, carcinoma cells and thyroid tumor cells produce this enzyme under all conditions examined.
A major hydrophilic form of AChE with the potential to be “tailed” by non-catalytic subunits is expressed in brain and muscle whereas a hydrophobic, phosphoinositide (PI)-linked form of the enzyme is found in erythrocytes. Two sublines of the human erythroleukemic K-562 cells were shown to express the PI-linked form of AChE, however, with different structural properties of the PI moiety. To reveal the molecular mechanisms underlying the heterogeneous tumorigenic expression of AChE, applicants initiated the investigation of alternative splicing in AChEmRNAs from different tumor cells.
Alternative splicing controls the generation of proteins with diverse properties from single genes through the alternate excision of intronic sequences from the nuclear precursors of the relevant mRNAs (Pre-mRNA). It is known to be cell type-, tissue- and/or developmental stage-specific and is considered as the principal mechanism controlling the site(s) and timing of expression and the properties of the resultant protein products from various genes.
Alternative exons encoding the C-terminal peptide in AChE were shown to provide the molecular origins for the amphiphilic (PI)-linked and the hydrophilic “tailed” form of AChE in Torpedo electric organ. The existence of corresponding alternative exons (Li et al., 1991) and homologous enzyme forms in mammals suggested that a similar mechanism may provide for the molecular polymorphism of human AChE. However, the only cDNAs reported to date from mammalian brain and muscle encode the hydrophilic AChE form (see Soreq et al., 1990). Nonetheless, RNA-protection and PCR analyses have demonstrated the existence of two rare alternative AChEmRNAs in mouse hemopoietic cells (Li et al., 1991).
More specifically, three alternative AChE-encoding mRNAs have been described in mammals. The dominant brain and muscle AChE found in NMJs (AChE-T) is encoded by an mRNA carrying exon E1 and the invariant coding exons E2, E3, and E4 spliced to alternative exon E6 (Li et al., 1991; Ben Aziz-Aloya et al., 1993). AChEmRNA bearing exons E1-4 and alternative exon E5 encodes the glycolipid phosphatidylinositol (GPT)-linked form of AChE characteristic of vertebrate erythrocytes (AChE-H) (Li et al., 1993; Legay et al., 1993a). An additional readthrough mRNA species retaining the intronic sequence I4 located immediately 3′ to exon E4 was reported in rodent bone marrow and erythroleukemic cells (Li et al., 1993; Legay et al., 1993a) and in various tumor cells lines of human origin (Karpel et al., 1994). The tissue-specific posttranscriptional and posttranslational management of AChEmRNA and its polypeptide products raised the question of whether alternative C-terminal peptides play a role in mediating the accumulation of AChE in NMJs. It would be useful to be able to control drug distribution in vivo by targeting the neuromuscular junction or at other cites involving cholenergic receptors.
AChE is accumulated at neuromuscular junctions (Salpeter 1967) where it serves a vital function in modulating cholinergic neurotransmission (Reviewed by Soreq and Zakut, 1993). The molecular mechanisms by which AChE and other synaptic proteins are targeted to the NMJ are poorly understood. Compartmentalized transcription and translation in and around the junctional nuclei probably contribute to the NMJ localization of AChE (Jasmin et al., 1993). However, the high concentration of AChE at NMJs suggests that an additional step(s) may be required to actively direct this molecule to its ultimate synaptic destination. In that case, it is possible to postulate the existence of a unique molecular “tag” identifying AChE as NMJ-bound. Applicant has suggested the possibility that an evolutionarily conserved NMJ-targeting signal is embedded within the primary amino acid sequence of the major brain and muscle form of AChE (Ben Aziz-Aloya et al., 1993) but its exact sequence and activity when isolated was not known.
Anti-cholinesterase drugs are employed to treat a variety of frequently occurring diseases including Alzheimer's and Parkinson's diseases, glaucoma, multiple sclerosis, and myasthenia gravis (reviewed in Millard and Broomfield, In press). As a brief summary, glaucoma is a leading cause of blindness. Several different kinds of glaucoma exist, but the most common is primary open-angle glaucoma (POAG). Because little is known conclusively about the etiology of this disease, present medical treatment is purely symptomatic. For at least thirty years, ophthalmologists have been treating advanced POAG with anti-ChE compounds. The most often-used has been echotiophate; other agents have included DFP, neostigmine, physostigmine, paraoxon and tetraethylpyrophosphate (TEPP).
Physostigmine was first reported to mitigate the autoimmune disease, myasthenia gravis (MG), and provided the basis of a diagnostic test that enabled detection of moderate forms of the disease. This work was the impetus for uncovering the cause of organophosphorus nerve agent toxicity and, sixty years later, quaternary carbamate compounds, such as neostigmine and pyridostigmine, are still used in the symptomatic treatment of MG to provide increased neuromuscular transmission and, to some extent, greater muscular strength. Edrophonium, a reversible anti-ChE, also improves MG by compensating for reduction of ACh receptors through elevation of neurotransmitter levels.
Senile demential of Alzheimer type (SDAT) is one of the most common causes of mental debilitation among the elderly. SDAT coincides with selective degeneration of basal forebrain cortical cholinergic neurons and “neurofibrillary tangles” contain both AChE and BuChE activity. Brain AChE activity apparently is reduced in SDAT. Several reports of specific reductions and increases in different brain AChE isoforms, as well as an abnormal SDAT-associated cerebrospinal fluid AChE isoelectric point variant have been reported. Because of the general destruction of normal presynaptic cholinergic fibersin SDAT, however, local changes in AChE may be quite distal to the cause of injury.
It has been suggested that a procedure to counter SDAT symptoms would be the inhibition of AChE to allow diffusion of ACh to become the rate limiting step of synaptic transmission and, hence, to conserve selectively the “functional” transmitter released. Thus, anti-ChEs would compensate for the diminished ACh released by the surviving cortical neurons. There was initial success in improving SDAT with arecoline and physostigmine but the latter was not sufficient to counteract completely the side-effects of inhibition. 1,2,3,4-tetraphydrop-9-aminoacridine (tacrine) has emerged as a candidate, but it is premature to conclude proof of efficacy and it is possible that it acts by stimulating ACh synthesis, as well as by inhibiting ChEs.
Furthermore, anti-cholinesterase poisons form a broad category of agricultural and household pesticides including organophosphorous and carbamide agents. Research and development directed toward the production of new specific, effective, low-toxicity drugs and insecticides are abundant. However, heretofore, no effective in vivo system has been developed which would allow for the rapid, effective and reliable screening of such anti-cholinesterase substances.